Phloroglucinol Derivatives with Protein Tyrosine Phosphatase 1B

Article pubs.acs.org/jnp

Phloroglucinol Derivatives with Protein Tyrosine Phosphatase 1B Inhibitory Activities from Eugenia jambolana Seeds Feifei Liu,† Tao Yuan,‡ Wei Liu,† Hang Ma,§ Navindra P. Seeram,§ Yuanyuan Li,† Li Xu,† Yu Mu,† Xueshi Huang,*,† and Liya Li*,† †

Institute of Microbial Pharmaceuticals, College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China ‡ Key Laboratory of Plant Resources and Chemistry of Arid Zone and State Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, People’s Republic of China § Bioactive Botanical Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, United States S Supporting Information *

ABSTRACT: Fifteen new phloroglucinol derivatives, jamunones A−O (1−8 and 10−16, respectively), along with one known analogue spiralisone C (9), were isolated from Eugenia jambolana seeds. Their structures were elucidated by detailed nuclear magnetic resonance and mass spectrometry spectroscopic data interpretation. Compounds 1−9, 11, 12, and 14− 16 inhibited protein tyrosine phosphatase 1B activity with IC50 values ranging from 0.42 to 3.2 μM.

Eugenia jambolana Lam., which is also known as “Jamun” or “Indian blackberry”, belongs to the plant family Myrtaceae.1,2 This species is native to India and is distributed in the Indian subcontinent, Southeast Asia, and East Africa.3,4 E. jambolana has been used traditionally to treat diabetes in folk medicine, and different parts of the plant, especially the seeds from its edible fruit, have been reported to regulate blood glucose levels.5−8 Despite numerous studies that have investigated the potential antidiabetic activities of E. jambolana seeds, there is limited knowledge regarding the bioactive constituents responsible for these effects. Previous phytochemical studies of E. jambolana seeds have identified lipids,9 terpenes,10,11 flavonoids,12,13 tannins,12 phenolic acids,14 and phenylpropanoids.9 Our group has also reported on the identification and structural elucidation of several α-glucosidase inhibitory hydrolyzable tannins from a butanol extract of the seeds.15 More recently, we reported on the in vivo antidiabetic effect of an ethyl acetate extract of E. jambolana fruit pulp and the identification of the bioactive triterpene constituents present therein.16 In ongoing research of this medicinal plant, we observed that an ethyl acetate extract of E. jambolana seeds inhibited the activity (IC50 = 1.9 ± 0.10 μg/mL) of protein tyrosine phosphatase 1B (PTP1B), a key enzyme involved in the insulin signal transduction pathway and a potential therapeutic target © 2017 American Chemical Society and American Society of Pharmacognosy

for type 2 diabetes.17−19 Bioassay-guided investigation of this extract led to the isolation and structural elucidation of 15 new phloroglucinol derivatives, jamunones A−O (1−8 and 10−16, respectively), along with one known analogue, spiralisone C (9). All isolates bear a chromanone skeleton with an alkyl side chain. Herein are reported the isolation, structural elucidation, and PTP1B inhibitory activities of these compounds.

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RESULTS AND DISCUSSION Jamunone A (1) was obtained as a yellow gum. A molecular formula of C26H36O5 with nine degrees of unsaturation was established on the basis of the high-resolution electrospray ionization mass spectrometry (HRESI-MS) (m/z 429.2639 [M + H]+) and 13C nuclear magnetic resonance (NMR) data (Table 1). The IR spectrum suggested the presence of hydroxy group (3294 cm−1), alkyl (2928 cm−1), conjugated carbonyl (1640 cm−1), and aromatic ring (1608 and 1506 cm−1) functionalities. The 1H NMR data [in CDCl3, 600 MHz (Table 1)] of 1 indicated the presence of a hydrogen-bonded phenol group (δH 11.95, s, OH-5), two singlet aromatic protons (δH 5.99, s, H-6; δH 5.93, s, H-8), three nonconjugated 1,2disubstituted double bonds (δH 5.34−5.42, 6H, m), two doubly Received: November 19, 2016 Published: January 30, 2017 544

DOI: 10.1021/acs.jnatprod.6b01073 J. Nat. Prod. 2017, 80, 544−550

Journal of Natural Products

Article

Compound 2 was shown to have the molecular formula C28H40O5 by HRESI-MS (m/z 455.2806 [M − H]−; calcd for C28H39O5, m/z 455.2797) and 13C NMR data. The UV, IR, and NMR spectroscopic data of 2 were similar to those of 1, indicating 2 is a homologue of 1. Analysis of the NMR data (Table 1) revealed that both aromatic protons in 1 were absent, and instead, two additional methyl groups (δH 2.06; δC 6.7, CH3-6; δH 2.05; δC 7.5, CH3-8) were present in 2 and substituted at C-6 and C-8. This observation was confirmed by the HMBC correlation signals from CH3-6 to C-5 (δC 158.9) and C-7 (δC 160.7) and from CH3-8 to C-7 (δC 160.7) and C10 (δC 154.2). The (Z,Z,Z)-8,11,14-heptadecatrienyl side chain was determined to be the same as in 1 on the basis of the HMBC and 13C NMR spectra.21,23 Similar to jamunone A, 2 was also a racemic mixture on the basis of its zero optical rotation. Thus, the structure of 2, jamunone B, was assigned as indicated. Compounds 3 and 4 were isolated as an inseparable mixture of two isomers in a 4:3 ratio, on the basis of the analysis of the NMR spectroscopic data. The molecular formulas of 3 and 4 were determined to be C27H38O5 from the HRESI-MS ion observed at m/z 465.2625 [M + Na]+ (calcd for C27H38O5Na, m/z 465.2611), differing from 2 by 14 mass units. The NMR spectroscopic data of 3 and 4 (Table 1) were almost identical and showed a close correlation with those of 2 except for a slight difference in the benzene ring moiety in each case. Detailed HSQC and HMBC data analyses revealed there was only one singlet methyl group apparent in both 3 (δC 6.6) and 4 (δC 7.4), instead of two in 2 (δC 6.7 and 7.5), suggesting 3 and 4 to be C-demethylated derivatives of 2 at the C-8 and C-6 positions, respectively. These conclusions were supported by the 13C NMR data (C-6, at δC 104.1 in 3, δC 96.1 in 4, and δC 102.9 in 2; C-8, at δC 95.3 in 3, δC 103.8, in 4, and δC 102.1 in 2) and confirmed by the HMBC correlations of H-8 (δH 5.91) with C-6, C-7 (δC 162.6), C-10 (δC 157.4), and C-9 (δC 102.4) in 3 and of H-6 (δH 5.97) with C-5 (δC 161.3), C-7 (δC 163.0), C-8, and C-9 (δC 102.5) in 4. Hence, the structures of 3 and 4 could be proposed, and these compounds were named jamunones C and D, respectively. The molecular formulas of jamunones E (5, C26H38O5), F (6, C28H42O5), G (7, C27H40O5), and H (8, C27H40O5) were established on the basis of HRESI-MS and 13C NMR data. Similar to compounds 3 and 4, compounds 7 and 8 were also isolated as a pair of isomers in a 3:2 ratio based on the NMR data. The UV, IR, and NMR spectroscopic data of 5−8 were similar to those of 1−4, indicating they are structural analogues with one less degree of unsaturation in each case. Detailed analysis of the NMR spectroscopic data of 5−8 (Table 2) suggested that the same lipid side chain moiety occurred in each of these four compounds, but that there were differences in the 2,5,7-trihydroxy-2,3-dihydrochromone moiety. Comparison of the NMR and MS data of 5−8 with those of 1−4 revealed that compounds 5−8 possess zero, two, and one methyl group at C-6 and/or C-8. The NMR spectroscopic data of the remaining lipid chain moiety of 5−8 closely resembled those of 1−4, except for the replacement of the double bond between C-14′ (δC 127.0−127.1) and C-15′ (δC 131.9−132.0) in 1−4 with two methylenes (δC 29.4 and 31.5) in 5−8. These observations were confirmed by the HMBC correlations from H3-17′ (δH 0.90 in 5 and 6 and δH 0.89 in 7 and 8) and H2-13′ (δH 2.08 in 5 and 6 and δH 2.06 in 7 and 8) to C-15′ and by supportive MS analysis. The Z geometry of each of the two double bonds in the common 8,11-heptadecadienyl side chain

allylic methylenes (δH 2.82, 4H, m), and a chain terminal methyl group (δH 0.99, t, H3-17′). The 13C NMR data [in CDCl3, 150 MHz (Table 1)] of 1 displayed 26 carbons, including a carbonyl (δC 194.9), six well-resolved olefinic carbons (δC 127.0, 127.7, 128.2 × 2, 130.1, and 131.9), six aromatic carbons (δC 95.9, 96.6, 102.5, 159.9, 163.8, and 164.4), one deshielded sp3 quaternary carbon (δC 102.3), and 12 aliphatic carbons (δC 14.2, 20.5, 23.1, 25.5, 25.6, 27.1, 29.0, 29.3 × 2, 29.5, and 41.1). All of the proton signals were assigned to the corresponding carbons through direct 1H and 13 C correlations in the heteronuclear single-quantum coherence (HSQC) spectrum. The spectroscopic data of 1 were closely related to those of jambone E, a known acylphloroglucinol derivative with a 5,7-dihydroxychromone skeleton, which was previously isolated from Syzygium jambos.20 Compared to those of jambone E, the resonances for the double bond between C-2 (δC 172.2) and C-3 (δH 5.95, s; δC 108.1) in the 1H and 13C NMR spectra were absent, and instead, a hemiketal carbon (δC 102.3, C-2) and a methylene carbon (δH 2.77, d, J = 16.9 Hz; δH 2.91, d, J = 16.9 Hz; δC 44.8, C-3) were present, indicating that jambone E is a dehydration derivative of 1. This conclusion was supported by the downfield shift of the carbonyl carbon at C-4 (δC 194.9 in 1 vs δC 183.7 in jambone E) and confirmed by the correlations of H2-3 with C-2 and C-4 in the heteronuclear multiple-bond correlation (HMBC) spectrum (Figure 1). The positions and Z geometry of each of the three double bonds in the heptadecatrienyl side chain were established on the basis of 1 H− 1 H correlation spectroscopy (COSY) and HMBC correlation analysis (Figure 1), and the carbon resonances for two doubly allylic methylenes were determined at δC 25.6 and 25.5 (C-10′ and C-13′, respectively).21−23 The zero optical rotation suggested that 1 occurred as a racemic mixture.21 Therefore, the structure of 1, assigned the trivial name of jamunone A, was determined as shown. 545



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Table 1. 1H NMR (600 MHz) and 13C NMR (150 MHz) Data for Compounds 1−4 in CDCl3 1 position

a

δC, type

2 3

102.3, C 44.8, CH2

4 5 6 7 8 9 10 Me-6 Me-8 1′ 2′ 3′a 4′a 5′a 6′a 7′ 8′ 9′ 10′a 11′ 12′ 13′a 14′ 15′ 16′ 17′ OH-5

194.9, C 163.8, C 96.6, CH 164.4, C 95.9, CH 102.5, C 159.9, C

41.1, CH2 23.1, CH2 29.5, CH2 29.3, CH2 29.3, CH2 29.0, CH2 27.1, CH2 130.1, CH 127.7, CH 25.6, CH2 128.2, CH 128.2, CH 25.5, CH2 127.0, CH 131.9, CH 20.5, CH2 14.2, CH3

2 δH [J (Hz)] 2.77, d (16.9) 2.91, d (16.9)

5.99, s 5.93, s

1.90, m 1.51, m 1.31−1.41, m 1.31−1.41, m 1.31−1.41, m 1.31−1.41, m 2.08, m 5.42, m 5.36, m 2.82, m 5.39, m 5.39, m 2.82, m 5.34, m 5.42, m 2.08, m 0.99, t (7.5) 11.95, s

δC, type 101.7, C 45.1, CH2 195.4, C 158.9, C 102.9, C 160.7, C 102.1, C 102.4, C 154.2, C 6.7, CH3 7.5, CH3 40.9, CH2 23.0, CH2 29.5, CH2 29.4, CH2 29.3, CH2 29.1, CH2 27.1, CH2 130.1, CH 127.7, CH 25.6, CH2 128.1, CH 128.2, CH 25.5, CH2 127.0, CH 131.9, CH 20.5, CH2 14.2, CH3

3 δH [J (Hz)] 2.77, d (16.8) 2.90, d (16.8)

2.06, s 2.05, s 1.92, m 1.54, m 1.32−1.42, m 1.32−1.42, m 1.32−1.42, m 1.32−1.42, m 2.06, m 5.41, m 5.34, m 2.82, m 5.39, m 5.39, m 2.82, m 5.34, m 5.42, m 2.09, m 0.98, t (7.6) 12.19, s

δC, type 102.1, C 44.9, CH2 195.1, C 161.4, C 104.1, C 162.6, C 95.3, CH 102.4, C 157.4, C 6.6, CH3 41.1, CH2 23.2, CH2 29.6, CH2 29.5, CH2 29.4, CH2 29.2, CH2 27.2, CH2 130.3, CH 127.8, CH 25.6, CH2 128.3, CH 128.3, CH 25.5, CH2 127.1, CH 132.0, CH 20.6, CH2 14.3, CH3

4 δH [J (Hz)] 2.74, d (16.3) 2.87, d (16.3)

5.91, s

δC, type 101.9, C 44.8, CH2 195.5, C 161.3, C 96.1, CH 163.0, C 103.8, C 102.5, C 156.9, C

δH [J (Hz)] 2.74, d (16.3) 2.87, d (16.3)

5.97, s

2.02, s 1.87, m 1.51, m 1.29−1.41, m 1.29−1.41, m 1.29−1.41, m 1.29−1.41, m 2.06, m 5.40, m 5.35, m 2.81, m 5.37, m 5.37, m 2.81, m 5.32, m 5.41, m 2.08, m 0.97, t (7.5) 12.18, s

7.4, CH3 41.0, CH2 23.1, CH2 29.6, CH2 29.5, CH2 29.4, CH2 29.2, CH2 27.2, CH2 130.2, CH 127.8, CH 25.6, CH2 128.2, CH 128.2, CH 25.5, CH2 127.1, CH 132.0, CH 20.6, CH2 14.3, CH3

2.00, s 1.92, m 1.51, m 1.29−1.41, m 1.29−1.41, m 1.29−1.41, m 1.29−1.41, m 2.06, m 5.40, m 5.35, m 2.81, m 5.37, m 5.37, m 2.81, m 5.32, m 5.41, m 2.08, m 0.97, t (7.5) 11.90, s

Assignments may be interchanged.

the HRES-IMS data. Furthermore, the pentadecanyl side chain found in 10−12 was determined to be similar to that in spiralisone C on the basis of NMR and MS data. Thus, the structures of compounds 10−12 (jamunones I−K, respectively) were determined as shown. Compounds 13−16 were assigned the molecular formulas C24H36O5, C26H40O5, and C25H38O5 (15 and 16), respectively, as determined from their HRESI-MS data (Table 4). Compounds 15 and 16 were also isolated as a pair of isomers in a 7:5 ratio based on the NMR data, similar to 3 and 4, 7 and 8, and 11 and 12. Comparison of the NMR data of compounds 13−16 with those of 9−12 revealed compounds 13−16 to be monounsaturated homologues of 9−12 with the replacement of the pentadecanyl side chain with a pentadecenyl side chain. This was deduced from the presence of two olefinic carbon signals (δC 129.6−130.1) in 13−16 instead of two methylenes (δC 29.2−29.6) in 9−12 and confirmed by detailed HMBC and MS data analysis. The position of the double bond in the C15 alkene chain of compounds 13−16 was determined via ESIMS/MS analysis. For compound 13, the fragment ion at m/z 405 [M + H − H2O]− was selected as the precursor ion for MS/MS analysis. Two diagnostic product ions at (a) m/z 345 and (b) m/z 289 originating from McLafferty rearrangement were observed in the MS/MS data (Figure S1), which allowed the assignment of the location of the double bond in 13 as being at Δ10′. The other two major product ions at m/z 205 and 153 corresponded to fragments c and d, respectively

Figure 1. Key 1H−1H COSY and HMBC correlations of compounds 2 and 5.

of 5−8 was determined on the basis of the 13C NMR resonance of the bisallylic methylene at 25.6−25.7 ppm.21−23 On the basis of the aforementioned data and consideration of the zero optical rotation data obtained for pure compounds 5 and 6, the structures of compounds 5−8 were proposed as shown. The NMR data of compounds 10−12 (Table 3) closely resembled those of the known compound spiralisone C (9),21 suggesting these four compounds are homologues of one another. Analysis of the HSQC and HMBC data revealed that 10−12 occurred as C-methylated derivatives of 9 at C-6 and/or C-8, similar to 2−4 versus 1 and 6−8 versus 5, as supported by 546



Article

Table 2. 1H NMR (600 MHz) and 13C NMR (150 MHz) Data for Compounds 5−8 in CDCl3 5 position

a

δC, type

2 3

102.3, C 44.8, CH2

4 5 6 7 8 9 10 Me-6 Me-8 1′ 2′ 3′a 4′a 5′a 6′a 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′a 15′ 16′ 17′ OH-5

194.9, C 163.8, C 96.6, CH 164.5, C 96.0, CH 102.5, C 160.0, C

41.1, CH2 23.1, CH2 29.5, CH2 29.3, CH2 29.3, CH2 29.1, CH2 27.1, CH2 129.9, CH 128.0, CH 25.6, CH2 127.8, CH 130.2, CH 27.1, CH2 29.4, CH2 31.5, CH2 22.5, CH2 14.0, CH3

6 δH [J (Hz)] 2.77, d (16.9) 2.91, d (16.9)

5.99, s 5.93, s

1.90, m 1.51, m 1.32−1.41, m 1.32−1.41, m 1.32−1.41, m 1.32−1.41, m 2.06, m 5.41, m 5.37, m 2.79, m 5.37, m 5.41, m 2.08, m 1.32−1.41, m 1.33, m 1.31, m 0.90, t (6.8) 11.95, s

7

δC, type 101.7, C 45.1, CH2 195.3, C 158.9, C 102.8, C 160.6, C 102.1, C 102.4, C 154.2, C 6.7, CH3 7.5, CH3 40.9, CH2 22.9, CH2 29.5, CH2 29.3, CH2 29.3, CH2 29.1, CH2 27.1, CH2 129.9, CH 127.8, CH 25.6, CH2 128.0, CH 130.2, CH 27.1, CH2 29.4, CH2 31.5, CH2 22.5, CH2 14.0, CH3

δH [J (Hz)]

δC, type 102.1, C 44.8, CH2

2.76, d (16.9) 2.89, d (16.9)

195.0, C 161.4, C 103.9, C 162.2, C 95.3, CH 102.4, C 157.4, C 6.6, CH3

2.07, s 2.05, s 1.93, m 1.54, m 1.33−1.42, m 1.33−1.42, m 1.33−1.42, m 1.33−1.42, m 2.08, m 5.41, m 5.37, m 2.79, m 5.37, m 5.41, m 2.08, m 1.33−1.42, m 1.32, m 1.31, m 0.90, t (6.9) 12.20, s

41.1, CH2 23.2, CH2 29.6, CH2 29.5, CH2 29.4, CH2 29.2, CH2 27.2, CH2 130.0, CH 127.9, CH 25.7, CH2 128.1, CH 130.3, CH 27.2, CH2 29.4, CH2 31.5, CH2 22.6, CH2 14.1, CH3

8 δH [J (Hz)] 2.75, d (16.3) 2.88, d (16.3)

5.91, s


δH [J (Hz)] 2.75, d (16.3) 2.88, d (16.3)

5.99, s

2.03, s 1.88, m 1.52, m 1.31−1.42, m 1.31−1.42, m 1.31−1.42, m 1.31−1.42, m 2.06, m 5.37, m 5.34, m 2.78, m 5.35, m 5.38, m 2.06, m 1.31−1.42, m 1.31, m 1.30, m 0.89, t (6.8) 12.21, s

7.4, CH3 41.0, CH2 23.0, CH2 29.6, CH2 29.5, CH2 29.4, CH2 29.2, CH2 27.2, CH2 130.0, CH 127.9, CH 25.7, CH2 128.1, CH 130.3, CH 27.2, CH2 29.4, CH2 31.5, CH2 22.6, CH2 14.1, CH3

2.01, s 1.93, m 1.52, m 1.31−1.42, m 1.31−1.42, m 1.31−1.42, m 1.31−1.42, m 2.06, m 5.37, m 5.34, m 2.78, m 5.35, m 5.38, m 2.06, m 1.31−1.42, m 1.31, m 1.30, m 0.89, t (6.8) 11.89, s

Signals overlapped, and the assignments may be interchanged.

(Figure S1). The Z geometry of the double bond was determined by the resonance of two allylic carbons (δC 27.1 and 27.2 for C-9′ and C-12′, respectively) in the 13C NMR spectrum.24 Considering the common biogenetic source of these compounds, the (Z)-10-pentadecenyl side chain was also assigned to compounds 14−16 in a manner similar to that of compound 13, using ESI-MS/MS and 13C NMR data analysis. The zero optical rotation of 13 and 14 suggested that these compounds also occurred as racemic mixtures. Hence, the structures of compounds 13−16 (jamunones L−O, respectively) were determined as depicted. Besides jamunones A−O, one known analogue, spiralisone C (9), was also isolated from the E. jambolana seed ethyl acetate extract. Acyl phloroglucinols make up a major class of secondary metabolites isolated from several natural sources, including members of the family Myrtaceae, with various bioactivities.22,25−27 Simple acyl phloroglucinols are assumed to be biosynthetic precursors of chromanones that undergo transformation into chromones via facile dehydration.21,28 To date, only 12 acyl phloroglucinols with 2-alkyl-chromanone skeletons have been reported from Antidesma membranaceum, Eriostemon rhomboideus, Polygonum aubertii, and Zonaria spiralis.21,28−30 This is the first report of 2-alkyl-chromanones from a plant in the Myrtaceae family. The inhibitory effects exhibited by the E. jambolana seed ethyl acetate extract against PTP1B activity were pursued by assaying isolates 1−9, 11, 12, and 14−16, along with the

positive controls 3-(3,5-dibromo-4-hydroxybenzoyl)-2-ethylbenzofuran-6-sulfonic acid-[4-(thiazol-2-ylsulfamyl)phenyl]amide (a known PTP1B inhibitor) and ursolic acid.31,32 The PTP1B inhibitory effects of compounds 3 and 4, 7 and 8, 11 and 12, and 15 and 16 were assayed as their isomeric pairs, while compounds 10 and 13 were not evaluated because of the limited quantities of these compounds available. As shown in Table 5, all of the samples tested displayed significant inhibitory effects against PTP1B, with IC50 values ranging from 0.42 to 3.2 μM, which were comparable to those of the positive controls.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on an Anton Paar MCP200 automatic polarimeter. Ultraviolet spectra were recorded with a Beckman Coulter DU 730 nucleic acid/protein analyzer. IR spectra were recorded with a Bruker Tensor 27 FT-IR spectrometer (film). One- and two-dimensional NMR spectra were recorded on a Bruker Advance III-600 MHz spectrometer (Bruker Co., Rheinstetten, Germany) with deuterated chloroform (CDCl3) or dimethyl sulfoxide (DMSO-d6) as the solvent. HRESI-MS experiments were performed using a Bruker Micro TOF-Q mass spectrometer (Bruker Daltonics, Billerica, MA). The PTP1B inhibitory assay was recorded on a SpectraMax M5 spectrophotometer (Molecular Devices, Sunnyvale, CA). Silica gel (100−200 mesh, 300− 400 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), Sephadex LH-20 (GE Healthcare Biosciences AB, Uppsala, Sweden), MCI gel (CHP-20P, Mitsubishi Chemical Corp., Tokyo, Japan), and ODS-A (S-50 μm, 12 nm, YMC Co., Ltd., Kyoto, Japan) were used for 547



Article

Table 3. 1H NMR (600 MHz) and 13C NMR (150 MHz) Data for Compounds 9−12a 9 position

δC, type

2 3

103.2, C 45.4, CH2

4 5 6 7 8 9 10 Me-6 Me-8 1′ 2′ 3′b 4′b 5′b 6′b 7′b 8′b 9′b 10′b 11′b 12′b 13′ 14′ 15′ OH-5

196.3, C 163.5, C 95.8, CH 167.2, C 96.1, CH 101.8, C 160.9, C

40.6, 23.6, 29.6, 29.5, 29.5, 29.5, 29.5, 29.5, 29.5, 29.4, 29.4, 29.2, 31.8, 22.6, 14.4,

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

10 δH [J (Hz)] 2.55, d (16.6) 3.02, d (16.6)

5.81, s 5.80, s

1.81, m 1.38, m 1.20−1.30, m 1.20−1.30, m 1.20−1.30, m 1.20−1.30, m 1.20−1.30, m 1.20−1.30, m 1.20−1.30, m 1.20−1.30, m 1.20−1.30, m 1.20−1.30, m 1.23, m 1.25, m 0.85, t (6.6) 12.03, s

δC, type 101.8, C 45.2, CH2 195.4, C 159.0, C 102.9, C 160.8, C 102.2, C 102.5, C 154.3, C 6.8, CH3 7.6, CH3 41.0, CH2 23.1, CH2 29.7, CH2 29.7, CH2 29.7, CH2 29.7, CH2 29.7, CH2 29.6, CH2 29.5, CH2 29.5, CH2 29.5, CH2 29.4, CH2 31.9, CH2 22.7, CH2 14.1, CH3

11 δH [J (Hz)]

2.77, d (17.0) 2.90, d (17.0)

2.06, s 2.04, s 1.92, m 1.54, m 1.23−1.41, m 1.23−1.41, m 1.23−1.41, m 1.23−1.41, m 1.23−1.41, m 1.23−1.41, m 1.23−1.41, m 1.23−1.41, m 1.23−1.41, m 1.23−1.41, m 1.26, m 1.29, m 0.88, t (6.8) 12.20, s

δC, type 102.6, C 45.6, CH2 196.2, C 160.8, C 103.1, C 165.4, C 95.4, CH 101.5, C 158.3, C 7.4, CH3 40.6, 23.6, 29.6, 29.5, 29.5, 29.5, 29.5, 29.5, 29.4, 29.4, 29.4, 29.2, 31.6, 22.6, 14.4,

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

12 δH [J (Hz)]

2.53, d (16.8) 2.99, d (16.8)

5.89, s


δH [J (Hz)] 2.53, d (16.8) 2.99, d (16.8)

5.91, s

1.86, s 1.81, m 1.39, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.24, m 1.25, m 0.85, t (6.6) 12.30, s

8.1, CH3 40.6, CH2 23.4, CH2 29.6, CH2 29.5, CH2 29.5, CH2 29.5, CH2 29.5, CH2 29.5, CH2 29.4, CH2 29.4, CH2 29.4, CH2 29.2, CH2 31.6, CH2 22.6, CH2 14.4, CH3

1.87, s 1.81, m 1.44, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.22−1.32, m 1.24, m 1.25, m 0.85, t (6.6) 11.98, s

a

Compounds 9, 11, and 12 were measured in DMSO-d6, and compound 10 was measured in CDCl3. bSignals overlapped, and the assignments may be interchanged.

column chromatography. Semipreparative HPLC was performed using an ODS column (250 mm × 10 mm, 5 μm, YMC-ODS-A). The known PTP1B inhibitor 3-(3,5-dibromo-4-hydroxybenzoyl)-2-ethylbenzofuran-6-sulfonic acid-[4-(thiazol-2-ylsulfamyl)phenyl]amide was purchased from Calbiochem (Shanghai, People’s Republic of China). Ursolic acid was previously isolated from our laboratory.16 Unless otherwise specified, all chemicals and solvents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shenyang, People’s Republic of China). Plant Material. The seeds of the E. jambolana fruit were collected in Gujarat, India, in July 2010 by Verdure Science (Noblesville, IN) and authenticated by L. Hingorani (Pharmanza, Gujarat, India) with an accompanying deposited voucher specimen (accession number EJLH1), as previously reported by our group.15 Extraction and Isolation. The dried seed powder of E. jambolana (5.5 kg) was successively extracted with petroleum ether (2 × 10 L) and a H2O/acetone solvent [30:70 (v/v), 4 × 10 L] at room temperature for 48 h. The combined 70% acetone extract was evaporated under reduced pressure to afford a dark brown residue (1.2 kg), which was suspended in H2O (2.5 L) and submitted to liquid− liquid partitioning with ethyl acetate [5 × 2.5 L (EtOAc)]. After solvent removal, the EtOAc extract (185.0 g) was subjected to a MCI gel CHP-20P column and eluted with a H2O/MeOH solvent [1:0 to 0:1 (v/v)] and finally with acetone, to yield seven fractions. The 100% MeOH eluted fraction (33.2 g) was chromatographed over a silica gel column [petroleum ether/acetone, 15:1 to 0:1 (v/v)] to yield 11 further fractions (fractions 1−11). Fraction 2 (2.2 g) was applied to a Sephadex LH-20 column eluting with MeOH to afford six subfractions (2.1−2.6). Subfraction 2.5 (92.6 mg) was separated on a silica gel column (CH2Cl2/EtOAc, 60:1) and further purified by semipreparative HPLC (MeOH/H2O, 93:7) to furnish compounds 2

(13.1 mg), 6 (13.4 mg), and 14 (6.0 mg). Subfraction 2.6 (12.2 mg) was separated by semipreparative HPLC (MeOH/H2O, 92:8) to yield 10 (2.7 mg). Fraction 3 (2.1 g) was loaded on a Sephadex LH-20 column with MeOH as the eluent to give four subfractions (3.1−3.4). Subfraction 3.2 (260 mg) was chromatographed on a silica gel column with CH2Cl2/EtOAc gradient mixtures (40:1 to 20:1), followed by separation with a series of semipreparative HPLC (MeOH/H2O, 94:6) steps, to give 1 (4.9 mg), a mixture of 3 and 4 (15.8 mg), 5 (8.3 mg), a mixture of 7 and 8 (6.7 mg), 9 (6.7 mg), a mixture of 11 and 12 (5.5 mg), 13 (2.1 mg), and a mixture of 15 and 16 (9.5 mg). ESI-MS/MS Analysis. Compounds 13−16 were analyzed on an Agilent (Santa Clara, CA) 1200 Infinity series system coupled to an Agilent 6420 triple quadrupole mass spectrometer equipped with a Turbo Ion Spray source, operating in positive ionization mode. Optimal values for the ESI-MS/MS parameters were as follows: 330 °C drying gas temperature, 10 L/min drying gas flow, 35 psi nebulizer pressure, 4000/−4000 V capillary voltage, m/z 10−500 scan range, 200 ms dwell time, and 30 eV collision energy. Compound 1. Colorless gum: [α]20 D 0 (c 1.0, MeOH); UVmax (MeOH) λmax (log ε) 332 (4.14), 290 (4.82) nm; IR (film) νmax 3294, 3011, 2928, 2855, 1640, 1608, 1506, 1464, 1399, 1329, 1161, 1066, 1010, 966, 886, 835 cm−1; for 1H NMR and 13C NMR data, see Table 1; HRESI-MS m/z 429.2639 [M + H]+ (calcd for C26H37O5, m/z 429.2641). Compound 2. Yellow gum: [α]D20 0 (c 1.0, MeOH); UVmax (MeOH) λmax (log ε) 348 (3.97), 296 (4.52) nm; IR (film) νmax 3394, 2928, 2855, 1715, 1636, 1461, 1364, 1298, 1123, 895, 811 cm−1; for 1H NMR and 13C NMR data, see Table 1; HRESI-MS m/z 455.2806 [M − H]− (calcd for C28H39O5, m/z 455.2803). Compounds 3 and 4. Off-white gum: UVmax (MeOH) λmax (log ε) 346 (4.13), 293 (4.64) nm; IR (film) νmax 3296, 3011, 2927, 2855, 548



Article

Table 4. 1H NMR (600 MHz) and 13C NMR (150 MHz) Data for Compounds 13−16a 13 position

δC, type

2 3

102.3, C 44.8, CH2

4 5 6 7 8 9 10 Me-6 Me-8 1′ 2′ 3′b 4′b 5′b 6′b 7′b 8′b 9′ 10′ 11′ 12′ 13′ 14′ 15′ OH-5

194.9, C 163.8, C 96.6, CH 164.5, C 96.0, CH 102.5, C 160.0, C

41.1, CH2 23.1, CH2 29.7, CH2 29.6, CH2 29.4, CH2 29.3, CH2 29.1, CH2 28.9, CH2 27.1, CH2 129.6, CH 130.0, CH 27.2, CH2 31.7, CH2 22.6, CH2 14.0, CH3

14 δH [J (Hz)]

2.77, d (16.7) 2.91, d (16.7)

5.99, s 5.93, s

1.90, m 1.51, m 1.30−1.40, m 1.30−1.40, m 1.30−1.40, m 1.30−1.40, m 1.30−1.40, m 1.30−1.40, m 1.30−1.40, m 5.37, m 5.37, m 2.03, m 1.29, m 1.27, m 0.89, t (6.8) 11.95, s

δC, type 101.7, C 45.1, CH2 195.3, C 158.9, C 102.9, C 160.7, C 102.1, C 102.4, C 154.2, C 6.7, CH3 7.5, CH3 40.9, CH2 22.9, CH2 29.7, CH2 29.6, CH2 29.4, CH2 29.3, CH2 29.1, CH2 28.9, CH2 27.1, CH2 129.6, CH 130.0, CH 27.2, CH2 31.7, CH2 22.6, CH2 14.0, CH3

15 δH [J (Hz)]

2.77, d (16.9) 2.90, d (16.9)

2.07, s 2.05, s 1.92, m 1.55, m 1.30−1.42, m 1.30−1.42, m 1.30−1.42, m 1.30−1.42, m 1.30−1.42, m 1.30−1.42, m 1.30−1.42, m 5.36, m 5.36, m 2.03, m 1.29, m 1.27, m 0.89, t (6.8) 12.20, s

δC, type 102.6, C 45.6, CH2 196.3, C 160.8, C 103.1, C 164.9, C 95.4, CH 101.6, C 158.3, C 7.4, CH3 40.6, CH2 23.6, CH2 29.6, CH2 29.6, CH2 29.5, CH2 29.3, CH2 29.0, CH2 28.8, CH2 27.1, CH2 130.1, CH 130.1, CH 27.1, CH2 31.6, CH2 22.6, CH2 14.4, CH3

16 δH [J (Hz)]

2.55, d (16.8) 2.99, d (16.8)

5.91, s


δH [J (Hz)] 2.53, d (16.8) 2.99, d (16.8)

5.93, s

1.86, s 1.82, m 1.38, m 1.24−1.33, m 1.24−1.33, m 1.24−1.33, m 1.24−1.33, m 1.24−1.33, m 1.24−1.33, m 1.24−1.33, m 5.33, m 5.33, m 1.99, m 1.24, m 1.26, m 0.85, t (6.8) 12.29, s

8.1, CH3 40.6, CH2 23.4, CH2 29.6, CH2 29.6, CH2 29.5, CH2 29.2, CH2 29.0, CH2 28.8, CH2 27.1, CH2 130.1, CH 130.1, CH 27.1, CH2 31.6, CH2 22.6, CH2 14.4, CH3

1.87, s 1.82, m 1.44, m 1.24−1.33, m 1.24−1.33, m 1.24−1.33, m 1.24−1.33, m 1.24−1.33, m 1.24−1.33, m 1.24−1.33, m 5.33, m 5.33, m 1.99, m 1.24, m 1.26, m 0.85, t (6.8) 11.97, s

a Compounds 13 and 14 were measured in CDCl3 and compounds 15 and 16 in DMSO-d6. bSignals overlapped, and the assignments may be interchanged.

2929, 2857, 1711, 1635, 1613, 1461, 1364, 1239, 1190, 1122, 937, 811 cm−1; for 1H NMR and 13C NMR data, see Table 2; HRESI-MS m/z 457.2950 [M − H]− (calcd for C28H41O5, m/z 457.2959). Compounds 7 and 8. Off-white gum: UVmax (MeOH) λmax (log ε) 344 (3.94), 293 (4.51) nm; IR (film) νmax 3313, 3010, 2927, 2856, 1639, 1609, 1498, 1462, 1329, 1188, 1161, 1109, 893 cm−1; for 1H NMR and 13C NMR data, see Table 2; HRESI-MS m/z 467.2756 [M + Na]+ (calcd for C27H40O5Na, m/z 467.2768). Compound 10. Light yellow gum: [α]20 D 0 (c 0.8, MeOH); UVmax (MeOH) λmax (log ε) 346 (4.23), 294 (4.69) nm; IR (film) νmax 3384, 2922, 2852, 1635, 1611, 1466, 1366, 1325, 1295, 1189, 1123, 896 cm−1; for 1H NMR and 13C NMR data, see Table 3; HRESI-MS m/z 433.2955 [M − H]− (calcd for C26H41O5, m/z 433.2594). Compounds 11 and 12. Off-white amorphous powder: UVmax (MeOH) λmax (log ε) 334 (3.93), 293 (4.58) nm; IR (film) νmax 3344, 2921, 2852, 1639, 1607, 1465, 1328, 1161, 1107, 891, 819 cm−1; for 1 H NMR and 13C NMR data, see Table 3; HRESI-MS m/z 419.2822 [M − H]− (calcd for C25H39O5, m/z 419.2797). Compound 13. Colorless gum: [α]20 D 0 (c 1.0, MeOH); UVmax (MeOH) λmax (log ε) 332 (4.06), 290 (4.52) nm; IR (film) νmax 3320, 2926, 2855, 1642, 1508, 1464, 1330, 1161, 1068, 1011, 885, 837 cm−1; for 1H NMR and 13C NMR data, see Table 4; HRESI-MS m/z 403.2497 [M − H]− (calcd for C24H35O5, m/z 403.2484); positive ESI-MS/MS [M + H − H2O ion]+ m/z 387.0, 345.0, 331.0, 317.0, 303.1, 289.0, 275.1, 260.9, 233.1, 218.9, 205.0, 178.9, 152.9, 138.9, 68.9, 57.0, 42.9. Compound 14. Light yellow gum: [α]20 D 0 (c 0.9, MeOH); UVmax (MeOH) λmax (log ε) 330 (3.88), 290 (4.41) nm; IR (film) νmax 3360, 2924, 2854, 1641, 1464, 1328, 1278, 1161, 1067, 886, 834 cm−1; for 1 H NMR and 13C NMR data, see Table 4; HRESI-MS m/z 431.2794 [M − H]− (calcd for C26H39O5, m/z 431.2797); positive ESI-MS/MS

Table 5. Inhibitory Effects of Compounds 1−9, 11, 12, and 14−16 against PTP1B

a

compound

IC50 (μM)

1 2 3/4 5 6 7/8 9 11/12 14 15/16 ursolic acida synthetic PTP1B inhibitora

0.45 ± 0.03 1.6 ± 0.09 1.3 ± 0.07 0.42 ± 0.03 2.7 ± 0.14 3.2 ± 0.17 2.0 ± 0.11 0.98 ± 0.06 1.8 ± 0.10 1.0 ± 0.06 8.9 ± 0.45 2.0 ± 0.54

Positive control substance.

1638, 1606, 1458, 1327, 1159, 1107, 891, 818 cm−1; for 1H NMR and 13 C NMR data, see Table 1; HRESI-MS m/z 465.2625 [M + Na]+ (calcd for C27H38O5Na, m/z 465.2611). Compound 5. Colorless gum: [α]20 D 0 (c 1.0, MeOH); UVmax (MeOH) λmax (log ε) 328 (3.90), 287 (4.42) nm; IR (film) νmax 3334, 3010, 2926, 2855, 1641, 1609, 1464, 1328, 1276, 1161, 1066, 1011, 885, 835, 722 cm−1; for 1H NMR and 13C NMR data, see Table 2; HRESI-MS m/z 429.2642 [M − H]− (calcd for C26H37O5, m/z 429.2641). Compound 6. Light yellow gum: [α]20 D 0 (c 1.0, MeOH); UVmax (MeOH) λmax (log ε) 350 (3.99), 296 (4.51) nm; IR (film) νmax 3389, 549



Article

[M + H − H2O ion]+ m/z 415.0, 373.3, 359.1, 345.0, 331.0, 317.0, 302.9, 288.7, 275.0, 260.8, 219.1, 205.0, 179.0, 153.0, 138.9, 69.2, 57.1, 43.1. Compounds 15 and 16. Off-white amorphous powder: UVmax (MeOH) λmax (log ε) 338 (3.73), 292 (4.28) nm; IR (film) νmax 3353, 2925, 2854, 1638, 1607, 1461, 1328, 1160, 1109, 889, 820 cm−1; for 1 H NMR and 13C NMR data, see Table 4; HRESI-MS m/z 419.2808 [M + H]+ (calcd for C25H39O5, m/z 419.2797); positive ESI-MS/MS [M + H − H2O ion]+ m/z 401.0, 359.0, 345.0, 331.0, 317.1, 303.2, 289.0, 274.7, 260.7, 219.0, 192.9, 176.8, 166.9, 152.9, 56.8, 43.0. PTP1B Inhibitory Activity Assay. Compounds 1−9, 11, 12, and 14−16 were evaluated for their inhibitory activity against PTP1B, according to a reported method with slight modification.31 3-(3,5dibromo-4-hydroxybenzoyl)-2-ethylbenzofuran-6-sulfonic acid-[4(thiazol-2-ylsulfamyl)phenyl]amide and ursolic acid were used as positive controls. A typical 100 μL enzymatic reaction system contained 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.3), 100 mM NaCl, 0.1% bovine serum albumin (BSA), 1 mM dithiothreitol (DTT), and 1.15 μg/mL recombined glutathione S-transferase (GST) PTP1B1-321. After incubation with samples for 10 min at 30 °C, the substrate p-nitrophenyl phosphate (pNPP) was added (final concentration of 2 mM) to initiate the reaction. The reaction was terminated by adding a 3 M NaOH aqueous solution after 30 min. All of the test samples and positive controls were prepared with dimethyl sulfoxide (DMSO) at a series of concentrations (final concentration of DMSO, 1%). The PTP1B inhibitory activity was determined by measuring the released p-nitrophenol, which exhibited absorbance at 405 nm. All samples were tested in triplicate. IC50 values were calculated by fitting data with Origin software (OriginLab Corporation, Northampton, MA).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01073. IR, MS, and NMR spectra of compounds 1−8 and 10− 16 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Telephone and fax: 0086-24-83656106. E-mail: huangxs@ mail.neu.edu.cn. *Telephone and fax: 0086-24-83656122. E-mail: [email protected]. edu.cn. ORCID

Xueshi Huang: 0000-0002-1561-8108 Liya Li: 0000-0002-1894-9500 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the National Natural Science Foundation of China (31401473, 81570788, and 81341102), Fundamental Research Funds for Northeastern University, People’s Republic of China (N142005002, N142002001, N120820002, and N130220001), and the Program for New Century Excellent Talents in Northeastern University. The plant material was kindly provided by Mr. Ajay Patel of Verdure Sciences (Noblesville, IN).

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REFERENCES

(1) Li, L.; Adams, L. S.; Chen, S.; Killian, C.; Ahmed, A.; Seeram, N. P. J. Agric. Food Chem. 2009, 57, 826−831. 550


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